Overview: Most effective vaccines in use today generate protective antigen-specific B cell memory. To be effective, memory B cells must target the right specificity, with sufficiently high affinity and express the appropriate antibody class t provide the host with long-term immune protection. These three cardinal attributes of antigen-specific B cell memory emerge progressively under the cognate guidance of follicular helper T (TFH) cells following initial priming and antigen re-challenge in vivo. While we know a great deal about circulating antibodies, still little is understood about the development of high-affinity clas-specific memory B cells that ultimately provides B cell-mediated immune protection in vivo. It is our long-term goal to unravel the cellular programs and molecular mechanisms that regulate the stepwise development of antigen-specific B cell memory in vivo. Research Focus: The production of high-affinity antigen-specific antibodies is one powerful means of targeting antigen clearance and is thought to be a major mechanism of action for all vaccines in use today. Upon exposure to foreign antigens, individual helper T (TH) cells and B cells are recruited into the immune response based on the individual specificity of their expressed antigen receptors. What follows is a sequential cascade of antigen-specific programming, functional differentiation and developmental re-programming that produces class switched, high-affinity memory B cells in ways that will be examined mechanistically in the current research proposal. Specific Aims: We will use a murine protein vaccination model to evaluate the changes in transcriptional programming that accompany stepwise antigen-specific memory B cell development. We propose that antigen recognition by specific B cells at different stages of development initiates separable transcriptional programs that mediate productive contact with antigen-specific TFH cells and propagate effective B cell memory. Here, we wil test multiple facets of this central hypothesis with particular focus on the regulation of affinity maturation in early germinal centers (SA-1), ongoing clonal evolution upon antigen recall (SA-2) and the programming and maintenance of antibody class (SA-3) as three defining attributes of antigen-specific B cell memory. Our studies seek to reveal B cell intrinsic mechanisms of memory B cel development that could define unique targets and new strategies to enhance the action and efficacy of future vaccines and targeted immunotherapies.